The use of acridinium esters as chemiluminescent labels in clinical assays is known. For example, European Patent Application No. 82306557.8 describes the use of an aryl acridinium ester linked to an N-succinimidyl moiety as a chemiluminescent label in immunoassays. U.S. Pat. No. 4,745,181 and copending U.S. patent application Ser. No. 133,792, filed Dec, 14, 1987, now U.S. Pat. No. 4,918,192 describe polysubstituted aryl acridinium esters which are useful in immunoassays and nucleic acid hybridization assays. U.S. patent application Ser. No. 826,186, filed Jan. 22, 1992, which is a continuation of Ser. No. 226,639, filed Aug. 1, 1988, describes hydrophilic polysubstituted aryl acridinium esters and conjugates thereof useful in clinical assays, particularly those assays involving liposomes.
Richardson et al (Clin. Chem. 31/10, 1664-1668, 1985) and Miller et al (Ann. Clin. Biochem 25, 27-34, 1988) describe the use of 4-(2-aminoethyl)-phenyl acridine-9-carboxylate in a chemiluminescent immunoassay for plasma progesterone.
However, the prior art acridinium esters often cannot effectively form conjugates with certain analytes. These analytes may lack nucleophilic groups or may contain carboxylic groups which are not readily amenable to modifications. In some cases, the nucleophilic group of the analyte cannot be acylated with an active-group containing acridinium ester because of the resulting deleterious effect on the immunoactivity of the analyte.
The acridine ester of Richardson et al and Miller et al cannot be converted to a useful acridinium ester without the concurrent loss of the nucleophilicity of the ester. The acridine ester must be conjugated first with the target analyte through a nucleophilic reaction, and then subsequently converted to an acridinium ester moiety. The general reaction conditions of the acridine ester-to-acridinium ester conversion are non-selective. As a result, susceptible groups on the target analytes are frequently affected, resulting in the loss of or reduction of the immunoactivity of the resulting conjugate.
Accordingly, it is the purpose of the present invention to provide novel nucleophilic polysubstituted aryl acridinium esters. It is also a purpose of the present invention to provide novel nucleophilic polysubstituted aryl acridinium esters which contain an additional ionizable group.
It is a further purpose of this invention to provide conjugates formed from the novel nucleophilic polysubstituted aryl acridinium esters.
It is a still further purpose of this invention to provide assays utilizing the novel nucleophilic polysubstituted aryl acridinium esters and conjugates thereof.